Formulations of bendamustine

ABSTRACT

Methods of treatment using bendamustine formulations designed for small volume intravenous administration are disclosed. The methods conveniently allow shorter administration time without the active ingredient coming out of solution as compared to presently available formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 14/857,064, filed Sep. 17, 2015, which is a continuation of U.S. application Ser. No. 14/714,578, filed May 18, 2015, now U.S. Pat. No. 9,144,568, issued Sep. 29, 2015, which is a continuation of U.S. application Ser. No. 13/838,090, filed Mar. 15, 2013, now U.S. Pat. No. 9,034,908, issued May 19, 2015, which in turn claims the benefit of priority from U.S. Provisional Patent Application Ser. Nos. 61/613,173, filed Mar. 20, 2012, and 61/669,889, filed Jul. 10, 2012, the disclosure of each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Bendamustine is used in the treatment of a number of cancers including leukemias, Hodgkin's disease and multiple myelomas. Bendamustine (present as the HCl salt) is the active ingredient of the commercial product Treanda™, a lyophilized powder for reconstitution. Current labeling requirements call for the reconstituted product to be immediately (within 30 minutes) diluted into 500 mL of parenterally acceptable diluents such as 0.9% saline (normal saline) or 2.5% dextrose/0.45% saline and administered as part of an intravenous infusion delivering 100 mg/m² over 30 minutes or 120 mg/m² over 60 minutes. The diluted admixture may be stored at 2-8° C. for up to 24 hours, or 3 hours at room temperature (15-30° C.); administration must be completed within this period due to limited chemical stability in aqueous solutions.

Solubility limitations at 2-8° C. with currently approved and/or available formulations are believed to prevent current formulations from being administered in smaller more concentrated infusion volumes up to about 150 ml; at volumes below 150 ml, solubility is not sufficient even at 25° C. Side effects associated with extravasation and local erythema, swelling and pain at the injection site also dictate that the infusion be as dilute as possible. Therefore, precautions are taken to avoid extravasation, including monitoring of the intravenous infusion site for redness, swelling, pain, infection, and necrosis during and after administration of bendamustine. Higher infusion volume and longer infusion times, however, are associated with many drawbacks. For example, after reconstitution, the current product has a short period of stability, degradation of the drug occurs from the time of reconstitution until the entire large volume infusion has been completely administered. The current label for Treanda™ therefore instructs that the admixture should be prepared as close as possible to the time of patient administration, and that administration of Treanda™ must be completed within the durations indicated above. From patient comfort and nursing administration points of view, higher infusion volumes and long infusion times are undesirable. Higher infusion volumes may be associated with higher likelihood of weight gain and edema. Shorter infusion times and smaller infusion volumes result in a better quality of life experience for the patient by reducing the overall “stress” to the patient and reducing the time spent in the infusion clinic. Shorter infusion times (and smaller volumes) also reduce the potential extravasation (and shorten the patient monitoring time required). It would be advantageous if the drug could be administered in smaller volumes and over shorter times. The present invention addresses these needs.

SUMMARY OF THE INVENTION

In a first aspect of the invention there are provided methods of treating or preventing cancer or malignant disease in a subject such as a human. The methods include parenterally administering a volume of about 325 ml or less of a liquid composition containing:

a) from about 0.05 to about 12.5 mg/ml of bendamustine or a pharmaceutically acceptable salt thereof;

b) a solubilizer comprising polyethylene glycol and propylene glycol; and optionally

c) a parenterally acceptable diluent;

over a substantially continuous period of less than or equal to about 30 minutes to a subject in need thereof.

In alternative aspects of the invention there are provided methods of treating or preventing a bendamustine-responsive condition in a subject such as a human. In a first embodiment the methods include administering less than or equal to 325 ml of a liquid composition which contains

Ingredient Concentration Range (mg/ml) Bendamustine HCl 0.05 to 1.6 Solubilizer 1 propylene glycol 0.30 to 6.5 Solubilizer 2 PEG 400  3.3 to 65 Monothioglycerol  0.02 to 0.35 NaOH  0.0 to 0.01 over a substantially continuous period of less than or equal to about 30 minutes to a subject in need thereof.

In a related second embodiment of this aspect of the invention, the methods include administering less than or equal to 325 ml of a liquid composition which contains

Ingredient Concentration Range (mg/ml) Bendamustine HCl 1.1 to 12.5 Solubilizer 1 propylene glycol 4.5 to 51  Solubilizer 2 PEG 400 45 to 500 Monothioglycerol 0.2 to 2.5  NaOH 0.0 to 0.04 over a substantially continuous period of less than or equal to about 30 minutes to a subject in need thereof.

The methods of the present invention take advantage of the fact that the concentration of the bendamustine HCl is below the room temperature solubility limit of the vehicle into which it is placed. As a result, the bendamustine does not precipitate during administration to the patient thereby substantially avoiding the side effects which would otherwise occur during small volume administration of therapeutic doses of the drug. In addition, patients or subjects with bendamustine-responsive conditions can be treated using substantially smaller parenteral volumes which are well below the standard 500 ml administration volume.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

In a first aspect of the invention there are provided methods of treating or preventing cancer or malignant disease in a subject or patient who is preferably a human. The methods generally include parenterally administering a volume of about 325 ml or less of a liquid composition containing:

a) from about 0.05 to about 12.5 mg/ml of bendamustine HCl or a pharmaceutically acceptable salt thereof, the HCl salt being preferred;

b) a solubilizer comprising polyethylene glycol and propylene glycol; and optionally

c) a parenterally acceptable diluent;

over a substantially continuous period of less than or equal to about 30 minutes to a subject in need thereof.

The solubilizer portion of the formulation preferably includes from about 0.3 to about 45% volume polyethylene glycol (PEG) and from about 0.03 to about 5% volume propylene glycol (PG), as calculated on the basis of the total or final volume administered. Stated alternatively, the final concentration of the PEG generally ranges from about 3 to about 500 mg/ml, while the final concentration of the PG generally ranges from about 0.5 to about 51 mg/ml. Within these general ranges, certain aspects of the invention include concentration ranges for the PEG of from about 45 to about 500 mg/ml or from about 3.3 to about 63.3 mg/ml; and for the PG ranges of from about 4.7 to about 50.6; or from about 0.02 to about 6.5 mg/ml.

In some aspects of the invention, the bendamustine is administered intravenously as part of an intravenous infusion. Contemplated infusion volumes are preferably less than 325 ml with volumes such as about 250 ml, 100 ml, and 50 ml, with each volume varying about +/−10% or +/−15% being preferred in some embodiments. In alternative aspects of the invention, the intravenous administration volume is suitable for IV bolus administration and may also include an amount of pharmaceutically acceptable diluent such as normal saline or one of the other diluents described herein which does not cause the solubility of the vehicle to fall below the concentration of the bendamustine. Stated alternatively, the final concentration of the bendamustine will be below the solubility of the combination vehicle containing the mixture of propylene glycol and PEG and diluent. While most aspects of the invention are described in the context of administering less than about 325 ml including all vehicle ingredients, excipients, etc., it should be appreciated that volumes as low as a few milliliters, e.g. about 2, can be used so long as the vehicle includes sufficient solubilizers to preserve the solubility of the bendamustine therein during administration to the patient.

For purposes of the present invention, the word “about” when used to modify infusion volumes or concentrations shall be understood to include values which may vary by amounts of about +/−10% or 15%. In certain embodiments where the infusion volume is about 50 ml, the concentration of the bendamustine HCl or other pharmaceutically acceptable salt thereof is preferably from about 0.5 to about 5.6 mg/ml. In embodiments where the infusion volume is about 100 ml, the concentration of the bendamustine HCl or other pharmaceutically acceptable salt thereof can be preferably from about 0.1 to about 3.2 mg/ml. Similarly, in some aspects of the invention where the infusion volume is about 250 ml, the concentration of the bendamustine HCl or other pharmaceutically acceptable salt thereof is from about 0.05 to about 1.4 mg/ml.

The solubilizer is preferably a mixture of polyethylene glycol, hereinafter “PEG” and propylene glycol, hereinafter “PG”. The solubilizer can also optionally include an antioxidant such as monothioglycerol. The amount of antioxidant included is a formulation stabilizing amount, which, in the case of monothioglycerol ranges from about 2 to about 10 mg/ml. The PEG preferably has a molecular weight of about 400, i.e. PEG 400. Other molecular weight PEG's known to those of ordinary skill can be included if desired in alternative embodiments.

Certain aspects of the invention call for the ratio of the PEG to PG found in the solubilizer to be about 90:10. In alternative aspects, the ratio of the PEG to PG is about 85:15.

In some aspects of the invention, the total amount of solubilizer, i.e. blend of PEG and PG, included in infusion volumes of about 100-115 ml is from about 0.5 to about 26.5% vol.; while amounts of from about 0.2 to about 5% vol. for the solubilizer are preferably included in infusion volumes of about 250-265 ml; with solubilizer amounts of from about 2.0 to about 22.4% vol. included in infusion volumes of about 50-65 ml.

Since the solubilizer is a blend, the amount of PEG and PG in various volumes (calculated as % vol.) can be as follows:

Solubilizer 50 ml 100 ml 250 ml PEG 20.12 11.33 4.9 PG 2.24 1.26 0.54

In some preferred embodiments, the methods of the invention are advantageously carried out using bendamustine HCl containing compositions administered as small volume infusions with volumes of about 50 ml or about 100 ml or about 250 ml. Such smaller volumes allow the drug to be administered over a time period of about 10 minutes or less as part of an intravenous infusions containing a volume of about 50 ml; about 15 minutes or less as part of an intravenous infusions containing a volume of about 100 ml or when volumes of about 250 ml are infused, the IV infusion is administered over a time period of about 30 minutes or less. Depending upon the amount of drug administered, the IV bolus volumes containing sufficient amount of the drug will be less than 50 ml, with amounts of about 10 or 15 to 30 ml being sufficient.

The infusible compositions in many aspects of the invention will also preferably include the parenterally acceptable diluents such as water for injection (WFI), 0.9% saline (normal saline, preferred), 0.45% saline (half normal saline) or 2.5% dextrose/0.45% saline. Formulations well suited for carrying out the methods described herein are also described in commonly assigned U.S. patent application Ser. No. 13/016,473, filed Jan. 28, 2011, and Ser. No. 13/767,672 filed Feb. 14, 2013, the contents of which are incorporated herein by reference. As reviewed in the '672 patent application, some preferred bendamustine formulations can also include a minor amount of a pH adjuster such as sodium formate, sodium phosphate, potassium hydroxide, phosphoric acid or, preferably, sodium hydroxide.

In an alternative embodiment of the invention, the bendamustine formulations used in the methods described herein can be one or more of those described in U.S. Pat. Nos. 8,344,006 and 8,076,366; and US Patent Application Nos. 2013/0041004; 2012/0071532; 2010/0216858; 2006/0159713; and 2013/0041003, the contents of each of which are incorporated herein by reference. It being understood that the vehicle into which the bendamustine HCl is placed will have sufficient bendamustine solubility which exceeds the concentration of the drug included therein.

If desired, a sufficient amount of a concentrated, ready to use liquid formulation such one containing 25 mg/ml bendamustine HCl and already admixed with sufficient solubilizers can be transferred to a suitable fixed volume diluent container such as a bag containing 50, 100, 250 ml normal saline or the like. Alternatively, lyophilized bendamustine HCl can be reconstituted, combined with sufficient solubilizer blends as described herein and administered in accordance with the inventive methods. In such embodiments, the actual amount delivered to the patient will be slightly more than the diluent amount so as to allow for the addition of the drug/solubilizer vehicle.

In some aspects of the invention, there are provided methods of treating or preventing chronic lymphocytic leukemia (CLL). The small volume infusions can be given as part of any treatment protocol for which bendamustine is included. Thus, the compositions described herein can be administered as part of a poly-pharmaceutical treatment regimen according to known protocols with the exception that the concentrated bendamustine compositions described herein are administered in smaller infusion volumes over significantly shorter administration periods. For example, some CLL treatment regimens can include administering the compositions described herein intravenously as part of about 100 ml infusions in about 20 minutes or less and more preferably in about 15 minutes or less on days 1 and 2 of a 28 day cycle and repeating the cycle up to 6 times or longer if clinically appropriate. If 250 ml volumes are used to deliver the bendamustine, the time of administration is preferably about 30 minutes or less. If 50 ml volumes are used to deliver the bendamustine, the time of administration is preferably about 10 minutes or less.

In spite of the smaller volumes, the amount of bendamustine HCl administered to the patient in need thereof per dose (infusion or otherwise) in some preferred embodiments is about 100 mg/m². In some alternative aspects of the invention, the amount of bendamustine HCl administered to the patient in need thereof as part of the 50, 100 or 250 ml infusion is an amount sufficient to provide a dosage of 50 or 25 mg/m². Additional administration dosages will be apparent to those of ordinary skill based upon clinical experience, patient need without undue experimentation.

In other aspects of the invention, there are methods of treating or preventing the malignant disease of indolent B-cell non-Hodgkin's lymphoma. In these aspects, the composition is administered intravenously as a 100 ml infusion in less than 20 minutes and more preferably in about 15 minutes or less on days 1 and 2 of a 21 day cycle for up to 8 cycles or longer if clinically appropriate. If 250 ml volumes are used to deliver the bendamustine, the time of administration is preferably about 30 minutes or less. If 50 ml volumes are used to deliver the bendamustine, the time of administration is preferably about 10 minutes or less. The amount of bendamustine administered to the subject is preferably about 120 mg/m², although in alternative embodiments, the amount administered ranges from about 90 or 60 mg/m². As will be appreciated, further alternative dosage amounts will be apparent to those of ordinary skill based upon clinical experience, patient need without undue experimentation.

It will be appreciated by those skilled in the art that the above-mentioned dosages calculated in mg/m² for purposes of body surface area (BSA) are consistent with the bendamustine HCl concentrations also described herein, e.g. 0.5 to 5.6 mg/ml. In the alternative, the invention also contemplates IV bolus administration of bendamustine-containing formulations in volumes which can be administered via syringe, e.g. from a few milliliters up to about 50 milliliters, with therapeutic amounts of the drug in a concentration which does not exceed the vehicle solubility for the drug therein.

Further embodiments of the invention include methods of treating or preventing a bendamustine-responsive condition in a subject such as a human. In a first embodiment, the methods include administering less than or equal to 325 ml of a liquid composition which contains

Ingredient Concentration Range (mg/ml) Bendamustine HCl 0.05 to 1.6 Solubilizer 1 propylene glycol 0.30 to 6.5 Solubilizer 2 PEG 400  3.3 to 65 Monothioglycerol  0.02 to 0.35 NaOH  0.0 to 0.01 over a substantially continuous period of less than or equal to about 30 minutes to a subject in need thereof. More preferably, the administration time is well below 30 minutes and the administration time will decrease as the volume administered decreases.

Bendamustine formulations containing the above ingredients are capable of delivering approximately 25 mg of the drug as the HCl salt in volumes of pharmaceutically acceptable diluent ranging from about 325 ml down to about 15 ml. For example, 1 ml of a bendamustine HCl ready to use liquid available from Eagle Pharmaceuticals containing

Ingredient Concentration (mg/ml) Bendamustine HCl 25 PG 103.2 PEG 400 1013.4 Monothioglycerol 5 NaOH 0.08 is combined with 300 ml of a normal saline diluent to provide a final IV infusion containing 301 ml and a bendamustine final concentration of 0.08 mg/ml.

One ml of the 25 mg/ml Eagle bendamustine HCl is diluted into additional diluent volumes as shown below:

Diluent Volume Final Volume Final Bendamustine (ml) (ml) Conc. (mg/ml) 200 201 0.12 100 101 0.25 50 51 0.49 30 31 0.81 15 16 1.56

The measured solubility of the bendamustine HCl in the diluent/solubilizer combination (50 ml diluent plus1 ml of 25 mg/ml bendamustine HCl and solubilizers, etc.) at room temperature was 10.5 mg/ml using normal saline and 14.2 mg/ml using half normal saline/dextrose. The solubility of the diluent/solubilizer combination far exceeded the bendamustine concentration, thus assuring the avoidance of precipitated drug prior to or during administration. As will be appreciated by those of ordinary skill, as the concentration of solubilizers increases with respect to the total volume in small administration doses, the solubility of the bendamustine is maintained.

In a related second embodiment of this aspect of the invention, the methods include administering less than or equal to 325 ml of a liquid composition which contains

Ingredient Concentration Range (mg/ml) Bendamustine HCl 1.1 to 12.5 Solubilizer 1 propylene glycol 4.5 to 51  Solubilizer 2 PEG 400 45 to 500 Monothioglycerol 0.2 to 2.5  NaOH 0.0 to 0.04 over a substantially continuous period of less than or equal to about 30 minutes to a subject in need thereof. As was the case above, the administration time will decrease with the decrease in volume administered.

Bendamustine formulations containing the above ingredients are capable of delivering approximately 360 mg of the drug as the HCl salt in volumes of pharmaceutically acceptable diluent ranging from about 325 ml down to about 15 ml. As was the case above, the measured solubility of the bendamustine HCl in the diluent/solubilizer combination (1 ml drug+solubilizers, etc. and 50 ml diluent) at room temperature was 10.5 mg/ml using normal saline and 14.2 mg/ml using half normal saline/dextrose.

Instead of using only 1 ml of the above described Eagle 25 mg/ml bendamustine HCl ready to use liquid, 14.4 ml is combined with various amounts of diluent.

Diluent Volume Final Volume Final Bendamustine (ml) (ml) Conc. (mg/ml) 300 314.4 1.15 200 214.4 1.68 100 114.4 3.15 50 64.4 5.59 30 44.4 8.11 15 29.4 12.24

In each case, the solubility of the diluent/solubilizer combination exceeds the bendamustine concentration, thus assuring the avoidance of precipitated drug prior to or during administration.

EXAMPLES

The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention.

Example 1

The solubility of bendamustine HCl, obtained from two different sources, in 0.9% saline and 0.9% saline containing from different amounts of a non-aqueous solubilizer comprising a mixture of polyethylene glycol 400 and propylene glycol (in the volume proportion of 90:10) with and without 5 mg/ml monothioglycerol was determined at both room temperature (22-23° C.) and at refrigerated temperature (5° C.). Essentially, an excess of bendamustine HCl was added to solvents comprising of various volume percent of the non-aqueous solubilizer in 0.9% saline, and allowed to equilibrate with shaking for 30 minutes at room temperature, or for 24 hours at refrigerated temperature. At the end of the equilibration step, the suspensions were filtered through a 0.2 micron filter to remove undissolved bendamustine, and the filtrate solutions analyzed for bendamustine HCl content using a HPLC assay; quantification was performed against a bendamustine HCl reference standard. The solubility data are presented in Table 1.

TABLE 1 Solubility of bendamustine HCl in 0.9% saline with various amounts of non-aqueous solubilizer (90:10 PEG400:PG with and without 5 mg/mL monothioglycerol (MTG)) Solubility of bendamustine HCl Volume Volume % (mg/mL) in 90:10 PEG 400/PG % of of Non- Room temperature 5° C. Dilution Normal aqueous API Source API Source API Source Fold Saline Solubilizer A B* B A n/a 100.0 0.0 3.461 3.304 1.175 40 97.5 2.5 3.987 3.889 nd 20 95.0 5.0 4.429 4.204 2.022 13.3 92.5 7.5 nd 4.742 nd 10 90.0 10.0 5.626 5.351 2.431 8 87.5 12.5 nd 5.825 nd 6.7 85.0 15.0 7.012 6.554 2.900 5.7 82.5 17.5 nd 7.641 3.328 5 80.0 20.0 8.642 8.492 3.824 3.3 70.0 30.0 12.006  11.407 nd *solvent also contained 5 mg/ml monothioglycerol nd = not determined; API = active pharmaceutical ingredient

Example 2

Bendamustine-containing compositions are prepared by adding 5 mg/ml of thioglycerol to a mixture containing 90% polyethylene glycol 400 and 10% propylene glycol. As indicated in the Table 2 below, NaOH may be added to the PEG in an amount sufficient to get apparent pH of greater than or equal to 6.5 as measured using the pH method outlined in the USP monograph for polyethylene glycol (PEG). Bendamustine (BDM) is then added to the sample to a concentration of 10 mg/ml.

TABLE 2 Formulation BDM - 10 mg/mL Thioglycerol - 5 mg/mL PEG 400:PG (90:10) qs to 1 mL BDM - 10 mg/mL Thioglycerol - 5 mg/mL PEG 400:PG (90:10) qs to 1 mL (PEG 400 Treated with NaOH)

The compositions are then admixed with normal saline based on the total dose of bendamustine HCl, which in turn is based on the patient body surface area (BSA) and the dosing regimen (100 mg/m² for CLL and 120 mg/m² for NLL; although dose modifications of 90, 60, 50, and 25 mg/m² are possible, only the highest two dosing regimens are considered for illustrative purposes, as these result in the highest concentration of bendamustine during infusion). The 100 ml infusion is then made by admixing the dose appropriate volume of the 10 mg/ml solution with a 100 ml portion of normal saline to provide an infusible composition containing the appropriate dose of bendamustine (as the HCl salt) in the final admixture, which can be administered intravenously over about 15 minutes to a patient in need thereof.

As seen in Table 3, the concentrations of bendamustine (as HCl salt) and the corresponding volume percent of non-aqueous component are well below the corresponding solubilities at both room temperature and refrigerated temperature as detailed in Table 1. For example, for a 2.0 m² (average) patient dosed at 120 mg/m2, the final concentration of bendamustine HCl in a 100 ml admixture is 1.94 mg/mL. This is above the solubility of bendamustine HCl at refrigerated storage conditions in the absence of any non-aqueous components (1.175 mg/ml as shown in Table 1 for 100% normal saline), as would be the case with the currently approved Treanda™ product, thereby precluding preparation and storage of a 100 ml admixture volume at refrigerated conditions. However, the use of the non-aqueous bendamustine formulation described in this example results in the presence of 19.4% of the non-aqueous component in the final admixture, which improves the solubility to about 3.8 mg/mL (solubility of 3.824 mg/mL at 2-8° C. with 20% non-aqueous component, as shown in Table 1). Therefore, the solubility with the non-aqueous formulation is well above the final concentration (of bendamustine HCl) of 1.94 mg/mL, allowing preparation and storage of the 100 ml admixture at refrigerated conditions. In this example, the room temperature solubilities in 100% normal saline and 80% normal saline (with 20% non-aqueous component) are about 3.3 mg/ml and 8.5 mg/ml, respectively (see Table 1), which are also well above the final concentration of 1.94 mg/ml. Therefore, 100 ml admixtures of the non-aqueous formulation described in the example may also be prepared and stored at room temperature. In addition, the non-aqueous formulation of bendamustine described in this example at Table 2 may be diluted into smaller infusion volumes ranging from 250 ml or less, and stored at either room temperature or refrigerated temperature, with bendamustine continuing to remain in solution for extended periods of time as compared to currently available formulations.

TABLE 3 Concentrations of bendamustine (BDM, as HCl salt) and corresponding volume % of non-aqueous (NA) component in the final admixture, for volumes ranging from 100 ml to 250 ml For 10 mg/mL Formulation 100 mg/m² dose 120 mg/m² dose 100 mg/m² dose 120 mg/m² dose BDM BDM BDM BDM BSA Conc % NA Conc % NA Conc % NA Conc % NA (m²) (mg/ml) comp. (mg/ml) comp. (mg/ml) comp. (mg/ml) comp. Admix. Volume 250 mL Admix. Volume 200 mL 1.00 0.38 3.8 0.46 4.6 0.48 4.8 0.57 5.7 1.25 0.48 4.8 0.57 5.7 0.59 5.9 0.70 7.0 1.50 0.57 5.7 0.67 6.7 0.70 7.0 0.83 8.3 1.75 0.65 6.5 0.77 7.7 0.80 8.0 0.95 9.5 2.00 0.74 7.4 0.88 8.8 0.91 9.1 1.07 10.7 2.25 0.83 8.3 0.97 9.7 1.01 10.1 1.19 11.9 2.50 0.91 9.1 1.07 10.7 1.11 11.1 1.30 13.0 2.75 0.99 9.9 1.17 11.7 1.21 12.1 1.42 14.2 3.00 1.07 10.7 1.26 12.6 1.30 13.0 1.53 15.3 Admix. Volume 150 mL Admix. Volume 100 mL 1.00 0.63 6.3 0.74 7.4 0.91 9.1 1.07 10.7 1.25 0.77 7.7 0.91 9.1 1.11 11.1 1.30 13.0 1.50 0.91 9.1 1.07 10.7 1.30 13.0 1.53 15.3 1.75 1.04 10.4 1.23 12.3 1.49 14.9 1.74 17.4 2.00 1.18 11.8 1.38 13.8 1.67 16.7 1.94 19.4 2.25 1.30 13.0 1.53 15.3 1.84 18.4 2.13 21.3 2.50 1.43 14.3 1.67 16.7 2.00 20.0 2.31 23.1 2.75 1.55 15.5 1.80 18.0 2.16 21.6 2.48 24.8 3.00 1.67 16.7 1.94 19.4 2.31 23.1 2.65 26.5

Example 3

The procedures of Example 2 are repeated except that the dose appropriate volume of the 10 mg/ml bendamustine solution is diluted into 250 ml of normal saline. The final concentration of bendamustine in the 250 ml volume container ranges from about 0.05 mg/ml to about 1.3 mg/ml.

Example 4

The approximately 100 ml bendamustine HCl infusion of Example 2 is administered to a patient in about 15 minutes.

Example 5

Bendamustine-containing compositions may be prepared by adding 5 mg/ml of thioglycerol to 90% polyethylene glycol 400 and 10% propylene glycol. As indicated in the Table 4 below, NaOH may be added in an amount sufficient to get apparent pH of greater than or equal to 6.5 as measured using the pH method outlined in the USP monograph for polyethylene glycol (PEG). Bendamustine is then added to the sample to a concentration of 25 mg/ml as indicated in Table 4 below.

TABLE 4 Formulation BDM -25 mg/mL Thioglycerol - 5 mg/mL PEG 400:PG (90:10) qs to 1 mL BDM - 25 mg/mL Thioglycerol - 5 mg/mL PEG 400:PG (90:10) qs to 1 mL (PEG 400 Treated with NaOH)

The compositions are then admixed with normal saline based on the total dose of bendamustine HCl, which in turn is based on the patient body surface area (BSA) and the dosing regimen (100 mg/m² for CLL and 120 mg/m2 for NLL; although dose modifications of 90, 60, 50, and 25 mg/m² are possible, only the highest two dosing regimens are considered for illustrative purposes, as these result in the highest concentration of bendamustine during infusion). Table 5 below provides the final concentration of bendamustine (as the HCl salt) in the final admixture, for volumes ranging from 250 ml to 50 ml.

As seen in Table 5, the concentrations of bendamustine (as HCl salt) and the corresponding volume percent of non-aqueous component are well below the corresponding solubilities at room temperature as detailed in Table 1, for all admixture volumes up to 50 ml. For example, for a 2.0 m² (average) patient dosed at 120 mg/m², the final concentration of bendamustine HCl in a 50 ml admixture is 4.03 mg/ml. This is above the solubility of bendamustine HCl at both refrigerated and room temperature conditions in the absence of any non-aqueous components (1.175 mg/ml at 2-8° C. and 3.304-3.461 mg/ml at room temperature, as shown in Table 1 for 100% normal saline), as would be the case with the currently approved Treanda product, thereby precluding preparation and storage of a 50 ml admixture volume. However, the use of the non-aqueous bendamustine formulation described in this example results in the presence of 16.1% of the non-aqueous component in the final admixture, which improves the room temperature solubility to about 6.5 mg/ml (solubility of 6.554 mg/ml and 7.012 mg/ml with 15% non-aqueous component, as shown in Table 1). Therefore, the solubility with the non-aqueous formulation is well above the final concentration (of bendamustine HCl) of 4.03 mg/mL, allowing preparation and storage of the 100 ml admixture at room temperature conditions. Therefore, the non-aqueous formulation of bendamustine described in this example may be diluted into smaller infusion volumes ranging from 250 ml or less, with bendamustine continuing to remain in solution if maintained at room temperature. However, at refrigerated temperatures, the concentrations of bendamustine (as HCl salt) and the corresponding volume percent of non-aqueous component exceed the corresponding solubilities as detailed in Table 1, for all admixture volumes equal to or below 150 ml. In the scenario above, the solubility at refrigerated conditions with 15% non-aqueous component has improved to 2.9 mg/ml but is still below the final concentration of 4.03 mg/ml. Therefore, 50 ml admixtures of the non-aqueous formulation described in the example cannot be prepared and stored at refrigerated temperatures. However, for a 150 ml admixture, the final concentration of bendamustine HCl in this scenario is 1.5 mg/ml with about 6.0% non-aqueous component, which is below the solubility limit (of 2.022 mg/ml at 5% non-aqueous at 2-8° C.). Therefore, the non-aqueous formulation of bendamustine described in this example may be diluted into smaller infusion volumes ranging from 250 ml to 50 ml, and stored at only room temperature (but not refrigerated temperature), with bendamustine continuing to remain in solution. For storage at refrigerated temperatures, the minimum admixture volume that can be used is 150 ml or higher.

TABLE 5 Concentrations of bendamustine (BDM, as HCl salt) and corresponding volume % of non-aqueous (NA) component in the final admixture, for volumes ranging from 100 ml to 250 ml For 25 mg/mL Formulation 100 mg/m² dose 120 mg/m² dose 100 mg/m² dose 120 mg/m² dose BDM BDM BDM BDM BSA Conc % NA Conc % NA Conc % NA Conc % NA (m²) (mg/ml) comp. (mg/ml) comp. (mg/ml) comp. (mg/ml) comp. Admix. Volume 250 mL Admix. Volume 200 mL 1.00 0.39 1.6 0.47 1.9 0.49 2.0 0.59 2.3 1.25 0.49 2.0 0.59 2.3 0.61 2.4 0.73 2.9 1.50 0.59 2.3 0.70 2.8 0.73 2.9 0.87 3.5 1.75 0.68 2.7 0.81 3.3 0.85 3.4 1.01 4.0 2.00 0.78 3.1 0.92 3.7 0.96 3.8 1.15 4.6 2.25 0.87 3.5 1.04 4.1 1.08 4.3 1.28 5.1 2.50 0.96 3.8 1.15 4.6 1.19 4.8 1.42 5.7 2.75 1.05 4.2 1.25 5.0 1.30 5.2 1.55 6.2 3.00 1.15 4.6 1.36 5.4 1.42 5.7 1.68 6.7 Admix. Volume 150 mL Admix. Volume 100 mL 1.00 0.65 2.6 0.78 3.1 0.96 3.8 1.15 4.6 1.25 0.81 3.2 0.96 3.8 1.19 4.8 1.42 5.7 1.50 0.96 3.8 1.15 4.6 1.42 5.7 1.68 6.7 1.75 1.11 4.5 1.33 5.3 1.64 6.5 1.94 7.7 2.00 1.27 5.1 1.50 6.0 1.85 7.4 2.19 8.8 2.25 1.42 5.7 1.68 6.7 2.06 8.3 2.44 9.7 2.50 1.56 6.3 1.85 7.4 2.27 9.1 2.68 10.7 2.75 1.71 6.8 2.02 8.1 2.48 9.9 2.92 11.7 3.00 1.85 7.4 2.19 8.8 2.68 10.7 3.15 12.6 100 mg/m² dose 120 mg/m² dose BSA BDM Conc % NA BDM Conc % NA (m²) (mg/ml) comp. (mg/ml) comp. Admix. Volume 50 mL 1.00 1.85 7.4 2.19 8.8 1.25 2.27 9.1 2.68 10.7 1.50 2.68 10.7 3.15 12.6 1.75 3.07 12.3 3.60 14.4 2.00 3.45 13.8 4.03 16.1 2.25 3.81 15.3 4.44 17.8 2.50 4.17 16.7 4.84 19.4 2.75 4.51 18.0 5.22 20.9 3.00 4.84 19.4 5.59 22.4

Example 6

The hemolytic potential of the non-aqueous bendamustine formulation indicated in Table 4 (Example 5), when admixed with 250 ml and 100 ml of normal saline, was assessed. The hemolysis study was conducted at the highest final bendamustine HCl concentrations expected at these admixture volumes, namely, for a 3.0 m² patient dosed at 120 mg/m². At this dosing, the final bendamustine HCl concentration for 250 ml and 100 ml admixture volumes is 1.36 mg/ml and 3.15 mg/ml, respectively (Table 5). Human whole blood (1 ml) was incubated at 37° C. for approximately 30 minutes with admixed bendamustine HCl solutions at 1.4 mg/ml or 3.2 mg/ml at blood to drug solution volumetric ratios of 1:2 and 1:1, respectively. These volumetric ratios correspond to infusion times of 15 minutes and 10 minutes, respectively, for the 250 ml and 100 ml admixture volumes. A placebo of the bendamustine formulation (without the active ingredient) was also evaluated at these concentrations and volumetric ratios. A positive control (1% saponin solution), a negative control (normal saline), and Treanda™ diluted in normal saline to the highest concentration stated in the prescribing information (0.6 mg/ml) were included in the study. Following incubation and centrifugation of the samples, the plasma was harvested and hemolysis was evaluated by spectrophotometric analysis for hemoglobin in the supernatant. The results are summarized in Table 6. No hemolysis was observed with the non-aqueous bendamustine formulation when diluted with saline at either concentration or volumetric (blood:drug solution) ratios, or with the corresponding placebo at comparable sample volumes; supernatants from all samples were light yellow. In conclusion, no hemolytic effects are observed with non-aqueous bendamustine formulations when diluted to smaller volumes (100 to 250 ml) and infused in shorter times (10-15 minutes) than current practice.

TABLE 6 Hemolytic Potential test results Hemoglobin^(a) Test Supernatant Tube Mixture (mg/dL) Result Color^(b) No. Test Article A (25 mg/mL, diluted to 3.2 mg/mL with saline) - 100 ml admixture Human Blood plus: 1 N Light yellow 1 0 N Light yellow 2 1 N Light yellow 3 Test Article A (25 mg/mL, diluted to 1.4 mg/mL with saline) - 250 ml admixture 2 N Light yellow 4 1 N Light yellow 5 1 N Light yellow 6 Test Article A Vehicle (Placebo, diluted with 100 ml saline) 0 N Light yellow 7 1 N Light yellow 8 0 N Light yellow 9 Test Article A Vehicle (Placebo, diluted with 250 ml saline) 1 N Light yellow 10 4 N Light yellow 11 1 N Light yellow 12 Treanda ™ (5 mg/mL, diluted to 0.6 mg/mL with saline) 3 N Light yellow 43 4 N Light yellow 44 2 N Light yellow 45 Negative Control (normal saline) 9 N Yellow 55 5 N Yellow 56 3 N Yellow 57 Positive Control (1% Saponin)^(c) 5949 P Red 58 5974 P Red 59 6386 P Red 60 N = Negative, no hemolysis. NA = Not applicable. P = Positive, hemolysis. ^(a)Hemoglobin index of the mixture supernatants. ^(b)Plasma separated from whole blood plasma. ^(c)1% Saponin. Saponin is a hemolytic agent used to lyse erythrocytes.

Example 7

The local tolerance (intravenous (IV) and perivascular (PV)) of the non-aqueous bendamustine-containing composition indicated in Table 4 (Example 5), when admixed with 100 ml of normal saline and infused over 10 minutes, was assessed. New Zealand White rabbits (3 males [IV] and 2 males [PV]) received a single dose of bendamustine formulation (admixed with 100 ml saline to a final concentration of 3.2 mg/ml bendamustine HCl) and corresponding placebo in the left and right ear, respectively. The formulation was administered as either intravenous infusion (5 mg/kg in 10 minutes), or perivascular injection (250 μl) to determine local tolerance. Treanda™ reconstituted and admixed with normal saline to a final concentration of 0.6 mg/ml (the highest concentration stated in the label) was also studied either as a 30 minute IV infusion (the shortest infusion time stated in the label), as well as perivascular injection (250 μl). Animals were held for a 96 hour (post-dose) observation period. During the observation period, dermal scores were recorded for all administration sites. At the end of the observation periods, animals were euthanized and a macroscopic and microscopic examination of both ears was performed. Parameters evaluated during the study were: viability, clinical observations, body weights, macroscopic observations and microscopic pathology.

The results of the local tolerance study are summarized in Table 7 (in life dermal observations) and Table 8 (microscopic pathology for perivascular administration).

In Life Dermal Observations:

As seen in Table 7, there was transient, dermal irritation in the form of slight to moderate erythema and moderate edema noted between 24 and 72 hours post dose, in each of the groups receiving either bendamustine-containing formulations or placebo material intravenously. At 96 hours, irritation was limited to a few individual sites treated with test or placebo articles. Only a limited number of animals were affected, and there was no consistent pattern of irritation within a dose group (either for test article or placebo). The bendamustine formulations were considered not to produce dermal irritation when administered intravenously.

Perivascular administration of bendamustine formulations (0.25 ml injection volume) produced dermal irritation in all groups. Local signs of dermal irritation following perivascular administration were mostly characterized by slight (group 6—Treanda™) or slight to moderate (group 7—non aqueous bendamustine formulation of example 5) erythema, and slight edema (groups 7). The severity of the irritation observed correlated with the dose and/or concentration of the test article administered, with placebo groups generally showing a lesser level of irritation than the corresponding test-article formulation.

TABLE 7 Summary of in life dermal observations Dermal observations during 96 hour post dose period (incidence and most severe level of erythema and edema noted) Left ear (Bendamustine- containing Right ear (Placebo Material - Left ear/Right ear formulation) material) Intravenous administration Group 1: Treanda ™ diluted to 0.6 — (2/3) Slight mg/ml in saline/Treanda Placebo - 500 ml admixture Group 2: Non-aqueous bendamustine — (1/3) Moderate formulation 25 mg/ml diluted to 3.2 mg/ml in saline/placebo (+saline) - 100 ml admixture Perivascular administration Group 6: Treanda ™ diluted to 0.6 (1/2) Slight (1/2) Slight mg/ml in saline/Treanda ™ Placebo - 500 ml admixture Group 7: Non-aqueous bendamustine (2/2) Moderate (2/2) Slight formulation 25 mg/ml diluted to 3.2 mg/ml in saline/placebo (+saline) - 100 ml admixture

Microscopic Pathology:

Intravenous administration of test articles/placebos was generally well tolerated; no test article related effects were observed. Perivascular administration of bendamustine-containing formulations (including Treanda™) was associated with dose and/or concentration related minimal to marked edema/collagen degeneration and mixed inflammation in perivascular tissues. The non-aqueous formulation of bendamustine (Group 7) was nominally more severe in grade than Treanda™ (Group 6).

TABLE 8 Incidence and Average Severity of Microscopic Findings at Perivascular Sites Treanda ™ Non Aqueous (diluted to Bendamustine 0.6 mg/ml)/ (diluted to Treanda ™ 3.2 mg/ml)/ Formulation Placebo Placebo Group number 6 7 No. Animals examined 2 2 RIGHT EAR (RE) - Placebo Incidence (Average Severity)* RE Injection site Hemorrhage 0 (0.0) 0 (0.0) Mixed Inflammation 0 (0.0) 0 (0.0) Edema/Collagen Degeneration 1 (0.5) 0 (0.0) Degeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) RE 2 cm distal Edema/Collagen Degeneration 0 (0.0) 0 (0.0) Degeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) RE 4 cm distal Degeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) LEFT EAR (LE) - Test Article Incidence (Average Severity) LE Injection site Hemorrhage 0 (0.0) 0 (0.0) Mixed inflammation 0 (0.0) 2 (2.0) Edema/Collagen Degeneration 1 (1.0) 2 (1.5) Epidermis, Crust/Pustule, 0 (0.0) 0 (0.0) Erosion/Ulceration Degeneration/Inflammation, Vascular 1 (1.5) 1 (1.5) LE 2 cm distal Hemorrhage 0 (0.0) 0 (0.0) Mixed Inflammation 1 (0.5) 1 (1.0) Edema/Collagen Degeneration 0 (0.0) 2 (2.0) Epidermis, Crust/Pustule, 0 (0.0) 0 (0.0) Erosion/Ulceration Degeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) LE 4 cm distal Hemorrhage 0 (0.0) 0 (0.0) Mixed inflammation 0 (0.0) 1 (1.5) Edema/Collagen Degeneration 0 (0.0) 1 (2.0) Epidermis, Crust/Pustule, 0 (0.0) 0 (0.0) Erosion/Ulceration Degeneration/Inflammation, Vascular 0 (0.0) 0 (0.0) *The number in parentheses represents the average severity score; the total of severity scores of the findings divided by the number of animals in the group.

Conclusion:

No test-article related irritation effects were observed for the non-aqueous formulation of bendamustine via the IV route, indicating that proper administration of this formulation did not result in any adverse local reaction. Perivascular administration of the non-aqueous bendamustine formulation, which is primarily related to effects that may occur if extravasation should occur, resulted in irritation that was generally comparable to Treanda. Therefore, the non-aqueous formulation of bendamustine described herein is well tolerated, despite the higher concentration of the smaller infusion volume preparation.

Example 8

The chemical stability of the non-aqueous bendamustine formulation (25 mg/ml) indicated in Table 4 (Example 5), when admixed with 50 ml and 100 ml of normal saline, was assessed. For each admixture volume, the admixture solutions were prepared at the expected lowest concentration (corresponding to a 1.0 m² patient dosed at 25 mg/m²) and the highest concentration (corresponding to a 3.0 m² patient dosed at 120 mg/m²) of bendamustine HCl in the final admixture. For the 50 ml admixture volume, the tested minimum and maximum concentrations are about 0.5 mg/ml and 6.0 mg/ml, respectively. For the 100 ml admixture volume, the tested minimum and maximum concentrations are about 0.25 mg/ml and 3.2 mg/ml, respectively. The chemical stability of Treanda™ was also determined at the lowest (0.2 mg/ml) and the highest (0.6 mg/ml) admixed concentrations stated in the label. The chemical stability was monitored at room temperature at periodic intervals up to 24 hours using a validated HPLC assay. The results are summarized in Table 9.

TABLE 9 Dilution (Admixed) Stability of Bendamustine Formulations in Normal Saline at Room Temperature Attribute Highest Concentration (3.2 mg/ml) Lowest Concentration (0.25 mg/ml) Time Initial 1 hr 3 hrs 6 hrs 24 hrs Initial 1 hr 3 hrs 6 hrs 24 hrs Formulation Non Aqueous Bendamustine Formulation 25 mg/ml admixed with 100 ml normal saline Assay (mg/ml) 3.155 3.090 3.060 3.085 2.895 0.240 0.234 0.229 0.224 0.196 Assay (% Initial) 100.0 97.9 97.0 97.8 91.8 100.0 97.5 95.4 93.3 81.7 Impurity - MCE (%) BLQ BLQ BLQ BLQ BLQ ND ND ND ND ND Impurity - HP1 (%) 0.244 0.606 1.237 2.236 6.707 0.525 1.449 3.495 5.529 13.424 Impurity - Dimer (%) BLQ BLQ BLQ 0.068 0.158 ND ND ND BLQ 0.063 Single unknown (%) 0.086 0.061 BLQ BLQ 0.098 ND ND ND ND BLQ Total (%) 0.33 0.67 1.24 2.30 6.96 0.58 1.45 3.50 5.53 13.49 Attribute Highest Concentration (6.4 mg/ml) Lowest Concentration (0.5 mg/ml) Time Initial 1 hr hrs 6 hrs 24 hrs Initial 1 hr 3 hrs 6 hrs 24 hrs Formulation Non Aqueous Bendamustine Formulation 25 mg/ml admixed with 50 ml normal saline Assay (mg/ml) 6.62 6.60 6.60 6.54 6.46 0.475 0.470 0.455 0.445 0.394 Assay (% Initial) 100.0 99.7 99.7 98.8 97.8 100.0 98.9 95.8 93.7 82.9 Impurity - MCE (%) BLQ BLQ BLQ BLQ 0.074 BLQ BLQ BLQ BLQ BLQ Impurity - HP1 (%) 0.137 0.265 0.528 0.945 2.967 0.567 1.618 3.719 5.892 14.427 Impurity - Dimer (%) BLQ BLQ BLQ 0.050 0.110 BLQ BLQ BLQ 0.065 0.115 Single unknown (%) 0.112 0.105 0.086 0.054 0.112 0.057 BLQ ND ND ND Total (%) 0.25 0.37 0.61 1.05 3.36 0.67 1.62 3.72 5.96 14.54 Attribute Highest Concentration (0.6 mg/ml) Lowest Concentration (0.2 mg/ml) Time Initial 1 hr 3 hrs 6 hrs 24 hrs Initial 1 hr 3 hrs 6 hrs 24 hrs Formulation Treanda ™ 5 mg/ml admixed with 500 ml normal saline Assay (mg/ml) 0.566 0.558 0.544 0.527 0.454 0.193 0.191 0.185 0.178 0.154 Assay (% Initial) 100.0 98.6 96.1 93.1 80.2 100.0 99.0 95.9 92.2 79.8 Impurity - MCE (%) 0.263 0.261 0.268 0.262 0.263 0.261 0.288 0.277 0.250 0.276 Impurity - HP1 (%) 1.250 2.248 4.730 7.287 16.887 1.231 2.241 4.770 7.462 17.504 Impurity - Dimer (%) 0.223 0.229 0.269 0.279 0.326 0.188 0.185 0.178 0.176 0.252 Single unknown (%) 0.103 0.103 0.101 0.097 0.081 0.077 0.079 0.103 0.083 0.066 Total (%) 1.97 2.97 5.50 8.04 17.66 1.85 3.01 5.41 8.07 18.27 MCE—monochloroethyl derivative; HP1—mono hydroxyl bendamustine

As shown in Table 9, Treanda™ when prepared as directed in the label (final concentration between 0.2-0.6 mg/ml) shows total degradation of about 5-6% in 3 hours at room temperature (corresponding to the room temperature stability claim in the label); monohydroxy bendamustine is the main degradant. In contrast, the non-aqueous bendamustine formulations admixed in either 50 ml or 100 ml saline show total degradation of less than 5-6% over 6 hours at the lowest concentrations tested, indicating that these admixtures are significantly less prone to degradation. This stabilizing effect is particularly pronounced at the higher concentrations (which are more typical), with chemical stability evident for 24 hours at these concentrations. The non-aqueous formulations of bendamustine thus offer better chemical stability than Treanda™ when admixed into smaller volumes. 

I claim:
 1. A method of treating chronic lymphocytic leukemia or indolent B-cell non-Hodgkin's lymphoma in a subject in need of treatment comprising: parenterally administering to the subject, over a period of less than or equal to about 15 minutes, about 100 ml or less of a liquid composition comprising: 0.96-5.59 mg/mL of bendamustine or a pharmaceutically acceptable salt thereof; 3.8-22.4 vol % of a nonaqueous component comprising polyethylene glycol and propylene glycol; a parenterally acceptable diluent; and optionally an antioxidant.
 2. The method of claim 1, wherein the subject is human.
 3. The method of claim 1, wherein the composition is administered in about 10 minutes.
 4. The method of claim 1, comprising administering about 50 ml of the composition.
 5. The method of claim 1, wherein the polyethylene glycol is PEG
 400. 6. The method of claim 1, wherein the weight ratio of polyethylene glycol to propylene glycol is about 90:10.
 7. The method of claim 1, wherein the antioxidant is monothioglycerol.
 8. The method of claim 1, for the treatment of chronic lymphocytic leukemia.
 9. The method of claim 1, for the treatment of indolent B-cell non-Hodgkin's lymphoma. 